Gfp mcherry tirf filter cube

For in vivo imaging, 10 μl cell suspension was placed on a 24 × 60–mm No. 1.5 coverslip and allowed to settle for ∼1–3 min. A ring of petroleum jelly or vacuum grease was added around the droplet and a 22 × 22–mm No. 1.5 coverslip with 5-μl drop of 10 mM HEPES, pH 7.4, and 5 mM EGTA was inverted onto the larger cover glass to form a sealed observation chamber. For TIRF imaging, a Nikon Eclipse Ti-U inverted microscope equipped with a 60×/1.49 numerical aperture (NA) TIRF objective and a through-the-objective TIRF illumination system was used. Excitation light was provided a 40-mW, 488-nm diode laser (Spectraphysics), filtered by a Nikon GFP/mCherry TIRF filter cube (Lechtreck, 2013 (link)), and images were recorded at 10 frames/s using an EMCCD camera (Andor iXon ×3 DU897) and the Elements software package (Nikon). Specimens were imaged at room temperature (24°C). ImageJ was used to analyze the videos and to extract still images, image averages, and kymograms. Photoshop (Adobe) was used to adjust brightness and contrast, and the figures were assembled in Illustrator (Adobe).
For electron microscopy, cells were fixed in glutaraldehyde and processed as previously described (Wilkerson et al., 1995 (link)). Images were collected using a JEOL JEM1011 electron microscope and processed as described above.

For in vivo imaging, a Nikon Eclipse Ti-U inverted microscope equipped with a 60×/1.49 numerical aperture (NA) TIRF objective and a through-the-objective TIRF illumination system was used. Excitation light was provided by 75-mW, 561-nm and 40-mW, 488-nm diode lasers (Spectraphysics), and filtered by a Nikon GFP/mCherry TIRF filter cube (Lechtreck, 2013 , 2016 ). The two-color emission was separated by using an image splitting device (Photometrics DualView2), and documented at 10 frames/s using an EMCCD camera (Andor iXon X3 DU897) and the Elements software package (Nikon). For photobleaching the entire flagellum, the laser intensity of the 488-nm laser was increased to 10% for 4–10 s. For photobleaching a specific area, a focused 488-nm laser beam was passed through the specimen in epifluorescence mode. To prepare the observation chamber, 10 μl of cells was placed on a 24 × 60 mm no. 1.5 coverslip previously applied with a ring of petroleum jelly. The cells were allowed to settle for ∼1–10 min, mixed with an equal volume of 10 mM HEPES and 6.25 mM EGTA (pH 7.4) under a 22 × 22 mm no. 1.5 coverslip. The images were analyzed and kymograms and walking averages were generated in FIJI (= ImageJ; National Institutes of Health). Merged images or kymograms were produced using Photoshop and figures were assembled in Illustrator (Adobe).